Peptides (v.27, #7)
Editorial Board (CO2).
Ghrelin in plants: What is the function of an appetite hormone in plants? by Suleyman Aydin; Hikmet Geckil; Fikriye Zengin; H. Ibrahim Ozercan; Fikret Karatas; Suna Aydin; Dilek Turgut-Balik; Yusuf Ozkan; Ferda Dagli; Venhar Celik (1597-1602).
In the present work, we provide compelling evidence for the expression of a ghrelin-like peptide hormone that has only been associated with animals, in various plant tissues. Ghrelin, the appetite stimulating hormone, has been identified from a number of different species including humans, rat, pig, mouse, gerbil, eel, goldfish, bullfrog and chicken. The study here was conducted using an immunohistochemistry assay to screen whether plants have any ghrelin immunoreactivity. In this respect, Prunus x domestica L. and Marus alba were examined. Immunohistochemistry results showed that there is a strong human ghrelin immunoreactivity substance in the parenchyma cells of these plants. This was entirely unexpected since this hormone was considered to be present solely in animals. Thus, this study is the first to report the presence of a peptide with ghrelin-like activity in plants, a finding that has only been observed in the animal kingdom. RIA analysis confirmed that these plants contain significant amounts of this substance. Furthermore, reverse-phase HPLC analyses of plant extracts showed an elution characteristic of the peptide identical to that of human ghrelin. In general, fruit from both plants had higher levels of the peptide than the vegetative parts.
Keywords: Ghrelin; Peptide hormones; Growth hormone release; Growth hormone secretagogue receptor;
Ghrelin gastrokinetic action in patients with neurogenic gastroparesis by M. Binn; C. Albert; A. Gougeon; H. Maerki; B. Coulie; M. Lemoyne; R. Rabasa Lhoret; C. Tomasetto; P. Poitras (1603-1606).
Ghrelin has been shown to accelerate gastric emptying in animals where its effect appeared mediated through the vagus nerve. We aimed to verify the gastrokinetic capacity of ghrelin in human. Patients with gastroparesis attributed to a neural dysregulation by diabetes (n = 5) or surgical vagotomy (n = 1) were evaluated. The emptying of a test meal (420 kcal) was determined by the C13 octanoic acid breath test. Saline or synthetic ghrelin 1–4 μg/kg were given in 1 min bolus at the end of the meal. T-lag and T-1/2 were shorter during ghrelin than during saline administration [33 ± 5 min versus 65 ± 14 min (p < 0.01) and 119 ± 6 min versus 173 ± 38 min (p < 0.001)]. Ghrelin injection therefore accelerated gastric emptying of a meal in humans even in presence of a deficient gastric innervation.
Keywords: Regulatory peptides; GI hormones; Motilin-related peptides; Diabetic gastroparesis; GI motility;
Peripheral ghrelin participates in the glucostatic signaling mediated by the ventromedial and lateral hypothalamus neurons by Andrew Solomon; Brant A. De Fanti; J. Alfredo Martínez (1607-1615).
Employing immunohistochemistry techniques, we examined the c-fos expression in different hypothalamic areas, when plasma glucose levels were modified by the administration of insulin and 2-deoxyglucose (2-DG) respectively. Subsequently, the hypoglycemia produced by an injection of insulin significantly increased feeding concomitant to higher c-fos expression in the arcuate nucleus (ARC), paraventricular nucleus (PVN), dorsomedial hypothalamus (DMH) and lateral hypothalamus (LH), while no statistical changes in the ventromedial hypothalamus (VMH) were found. Also, the glucopenia induced by 2-DG administration produced similar stimulatory effects on appetite and the neuronal activity affecting all the hypothalamic areas studied, including the VMH. The peripheral blockade of the orexigenic hormone ghrelin with a specific antibody (AGA) significantly decreased food intake as induced from acute hypoglycemia and glucopenia. Curiously, the conjoint AGA and insulin or 2-DG administration produced a differential effect on the hypothalamic neurons analyzed, by increasing the number of c-fos positive neurons in the ARC, PVN and DMH, but not in the VMH and LH. This outcome suggests an interactive effect of the glucostatic pathways involving these two areas with the ghrelin signaling.
Keywords: Ghrelin; Appetite; Glycemia; Hypothalamus; Insulin; 2-Deoxyglucose;
Inotropic and lusitropic effects of ghrelin and their modulation by the endocardial endothelium, NO, prostaglandins, GHS-R1a and KCa channels by João-Bruno Soares; Amândio Rocha-Sousa; Paulo Castro-Chaves; Tiago Henriques-Coelho; Adelino F. Leite-Moreira (1616-1623).
Contractile effects of ghrelin (10−9 to 10−6 M) were tested in rat papillary muscles of normal (n = 50) and hypertrophic (n = 16) right ventricles (RV). RV hypertrophy was induced by pulmonary hypertension using monocrotaline. In normal muscles, ghrelin was added either alone (n = 9) or after pre-treatment with indomethacin (cycloxygenase inhibitor, 10−5 M; n = 10), l-nitro-l-arginin (NO synthase inhibitor, 10−4 M; n = 9), d-Lys(3)-GHRP-6 (GHS-R1a antagonist; 10−4 M; n = 8) or apamin + charybdotoxin (KCa channels blockers; 10−6 M, n = 7), as well as after damaging the endocardial endothelium (n = 7). In hypertrophic muscles, ghrelin was added either alone (n = 9) or after pre-treatment with apamin + charybdotoxin (10−6 M, n = 7). Ghrelin concentration-dependently decreased active tension (AT) and maximal velocity of tension rise (negative inotropic effect), as well as, maximal velocity of tension decay (negative lusitropic effect) and time to AT (onset of relaxation). These effects were maximal at 10−6 M, similar in normal and hypertrophic muscles and were significantly altered only by apamin + charybdotoxin, indomethacin and l-nitro-l-arginin. Apamin + charybdotoxin attenuated the negative inotropic effect, while indomethacin and l-nitro-l-arginin, respectively, blunted and exacerbated the premature onset of relaxation. In conclusion, ghrelin induces negative inotropic and lusitropic effects and an earlier onset of relaxation in normal and hypertrophic myocardium, which are independent of GHS-R1a, since they were not affected by d-Lys(3)-GHRP-6. The negative inotropic effect is partly mediated by KCa channels, while the earlier onset of relaxation is modulated by prostaglandins and NO.
Keywords: Ghrelin; Myocardium; GHS-R1a; Ca2+-activated K+ channels; NO; Prostaglandins;
Ghrelin stimulates food intake and growth hormone release in rats with thermal injury: Synthesis of ghrelin by Ambikaipakan Balasubramaniam; Steve Wood; Rashika Joshi; Chunhua Su; Lou Ann Friend; Sulaiman Sheriff; J. Howard James (1624-1631).
Ghrelin, a 28-residue octanoylated peptide recently isolated from the stomach, exhibits anti-cachectic properties through regulating food intake, energy expenditure, adiposity, growth hormone secretion and immune response. Burn injury induces persistent hypermetabolism and muscle wasting. We therefore hypothesized that ghrelin may also play a role in the pathophysiology of burn-induced cachexia. Overall ghrelin expression in the stomach over 10 days after burn was significantly decreased (p = 0.0003). Total plasma ghrelin was reduced 1 day after burn. Thus, changes in ghrelin synthesis and release may contribute to burn-induced dysfunctions. Ghrelin (30 nmol/rat, i.p.) greatly stimulated 2 h food intake in rats on five separate days after burn and in control rats. On post-burn day 15, plasma growth hormone levels were significantly lower than in controls, and this was restored to normal levels by ghrelin (10 nmol/rat, i.p.). These observations suggest that ghrelin retains its ability to favorably modulate both the peripheral anabolic and the central orexigenic signals, even after thermal injury despite ongoing changes due to prolonged and profound hypermetabolism, suggesting that long-term treatment with ghrelin may attenuate burn-induced dysfunctions.
Keywords: Burn injury; Food intake; Ghrelin; Growth hormone; Solid phase synthesis;
[Trp3, Arg5]-ghrelin(1–5) stimulates growth hormone secretion and food intake via growth hormone secretagogue (GHS) receptor by Kousaku Ohinata; Kanako Kobayashi; Masaaki Yoshikawa (1632-1637).
Ghrelin, a 28 amino acid peptide identified as an endogenous ligand for growth hormone secretagogue (GHS) receptor, stimulates food intake and growth hormone (GH) secretion. We designed low molecular weight peptides with affinity for the GHS receptor based on the primary structure of ghrelin. We found that [Trp3, Arg5]-ghrelin(1–5) (GSWFR), a novel pentapeptide composed of all l-amino acids, had affinity for the GHS receptor (IC50 = 10 μM). GSWFR stimulated GH secretion after intravenous or oral administration. Centrally administered GSWFR increased food intake in non-fasted mice. The orexigenic action of GSWFR was inhibited by a GHS receptor antagonist, [d-Lys3]-GH-releasing peptide-6, suggesting that GSWFR stimulated food intake through the GHS receptor. The orexigenic action of GSWFR was also inhibited by a neuropeptide Y (NPY) Y1 receptor antagonist, BIBO3304. These results suggest that the GSWFR-induced feeding is mediated by the NPY Y1 receptor.
Keywords: Ghrelin; Growth hormone secretagogue (GHS) receptor; Food intake; Growth hormone;
Effect of fatty acid chain length on suppression of ghrelin and stimulation of PYY, GLP-2 and PP secretion in healthy men by Kate L. Feltrin; Michael Patterson; Mohammad A. Ghatei; Stephen R. Bloom; James H. Meyer; Michael Horowitz; Christine Feinle-Bisset (1638-1643).
We have evaluated the effects of fatty acid chain length on ghrelin, peptide YY (PYY), glucagon-like peptide-2 (GLP-2) and pancreatic polypeptide (PP) secretion and hypothesized that intraduodenal administration of dodecanoic (“C12”), but not decanoic (“C10”), acid would decrease plasma ghrelin and increase PYY, GLP-2 and PP concentrations. Plasma hormone concentrations were measured in seven healthy men during 90-min intraduodenal infusions of: (i) C12, (ii) C10 or (iii) control (rate: 2 ml/min, 0.375 kcal/min for C12/C10) and after a buffet-meal consumed following the infusion. C12 markedly suppressed plasma ghrelin and increased both PYY and GLP-2 (all P < 0.05) compared with control and C10, while C10 had no effect. Both C10 and C12 increased PP concentrations slightly (P < 0.05). We conclude that the effects of intraduodenal fatty acids on ghrelin, PYY and GLP-2 secretion are dependent on their chain length.
Keywords: Fatty acid chain length; Ghrelin; Peptide YY; Glucagon-like peptide-2; Pancreatic polypeptide;
Neuropeptide Y administration into the amygdala alters high fat food intake by Stefany D. Primeaux; David A. York; George A. Bray (1644-1651).
The orexigenic effects of neuropeptide Y (NPY) are mediated through the hypothalamus, while the anxiolytic effects of NPY appear to be mediated through the amygdala. We hypothesized that intra-amygdalar administration of NPY might alter food preference without changing total food intake. Neuropeptide Y was administered into the central nucleus of the amygdala in both satiated and overnight-fasted rats, and intake and preference for a high fat diet (56%)/low carbohydrate (20%) diet or a low fat (10%)/high carbohydrate (66%) diet were measured. Intra-amygdalar NPY administration in satiated rats did not change total caloric intake, but it did produce a dose-dependent decrease in intake of and preference for high fat diet relative to low fat diet over 24 h. In overnight-fasted rats, intra-amygdalar NPY also decreased the intake and preference for a high fat diet relative to low fat diet over 24 h, without altering total caloric intake. Intra-amygdalar NPY administration did not produce conditioned taste aversions to a novel saccharin solution. These results suggest that amygdalar NPY may have a role in macronutrient selection, without altering total caloric intake.
Keywords: Neuropeptide Y; Amygdala; Food intake; Fat preference;
Chronic leptin infusion advances, and immunoneutralization of leptin postpones puberty onset in normally fed and feed restricted female rats by S. Zeinoaldini; J.J.M. Swarts; B.J.M. Van de Heijning (1652-1658).
Does leptin play a vital role in initiating puberty in female rats and can it overrule a nutrionally imposed (i.e. a 30% feed restriction, FR) delay in puberty onset? Prepubertal female rats were chronically infused for 14 days with leptin (icv or sc) or leptin-antiserum (icv) while puberty onset was monitored by means of scoring the moment of vaginal opening (VO). Median VO age was higher (35 days versus 27 days) in FR animals but leptin levels at VO were significantly decreased (1.44 ± 0.17 ng/ml versus 2.79 ± 0.31 ng/ml). Centrally (icv) and peripherally (sc) infused leptin (1 μg/day) advanced VO age compared to FR controls (30 days versus 35 days and 31 days versus 41 days, respectively). Congruently, centrally (icv) administered leptin-antiserum (0.6 μg/day) delayed puberty onset. In normally fed rats median VO age was only marginally advanced (26 days versus 27 days) but only if leptin was applied centrally. The effects of FR on puberty onset are counteracted or even normalized by the infusion of leptin, whereas immunoneutralization of central leptin postpones puberty onset. We therefore conclude that central leptin is crucial for initiating puberty in female rats.
Keywords: Intracerebroventricular; Immunoneutralization;
Haplotypes in the urotensin II gene and urotensin II receptor gene are associated with insulin resistance and impaired glucose tolerance by Kwok Leung Ong; Louisa Y.F. Wong; Yu Bun Man; Raymond Y.H. Leung; You-Qiang Song; Karen S.L. Lam; Bernard M.Y. Cheung (1659-1667).
We studied single nucleotide polymorphisms (SNPs) and haplotypes in the urotensin-II (UTS2) and urotensin-II receptor gene (UTS2R) in Hong Kong Chinese (224 hypertensive and 306 normotensive unrelated subjects) and their relation to hypertension and the metabolic syndrome. For UTS2, the GGT haplotype (−605G, 143G and 3836T) was associated with higher plasma level of U-II and insulin, and higher homeostasis model assessment of insulin resistance index and β-cell function. For UTS2R, the AC haplotype (−11640A and −8515C) was associated with higher 2 h plasma glucose after a 75 g oral glucose load. Therefore, U-II and its receptor may play a role in insulin resistance.
Keywords: Urotensin II; Hypertension; Insulin resistance; Metabolic syndrome; Single nucleotide polymorphism; Haplotype;
Potent anti-HIV activity of scytovirin domain 1 peptide by Changyun Xiong; Barry R. O’Keefe; R. Andrew Byrd; James B. McMahon (1668-1675).
Scytovirin (SVN) is a novel anti-HIV protein isolated from aqueous extracts of the cultured cyanobacterium Scytonema varium. SVN contains two apparent domains, one comprising amino acids 1–48 and the second stretching from amino acids 49 to 95. These two domains display significant homology to each other and a similar pattern of disulfide bonds. Two DNA constructs encoding scytovirin 1–48 (Cys7Ser) (SD1) and 49–95 (Cys55Ser) (SD2) were constructed, and expressed in E. coli, with thioredoxin fused to their N-terminus. Purified recombinant products were tested for binding activities with the HIV surface envelope glycoproteins gp120 and gp41. Whole cell anti-HIV data showed that SD1 had similar anti-HIV activity to the full-length SVN, whereas SD2 had significantly less anti-HIV activity. Further deletion mutants of the SD1 domain (SVN(3–45)Cys7Ser, SVN(6–45)Cys7Ser, SVN(11–45)Cys7Ser) showed that the N-terminal residues are necessary for full anti-HIV activity of SD1 and that an eight amino acid deletion from the C-terminus (SVN(1–40)Cys7Ser) had a significant effect, decreasing the anti-HIV activity of SD1 by approximately five-fold.
Keywords: Scytovirin; SD1; SD2; Microbicide; HIV; Anti-viral;
NMR structure of the viral peptide linked to the genome (VPg) of poliovirus by Catherine H. Schein; Numan Oezguen; David E. Volk; Ravindranath Garimella; Aniko Paul; Werner Braun (1676-1684).
VPgs are essential for replication of picornaviruses, which cause diseases such as poliomyelitis, foot and mouth disease, and the common cold. VPg in infected cells is covalently linked to the 5′ end of the viral RNA, or, in a uridylylated form, free in the cytoplasm. We show here the first solution structure for a picornaviral VPg, that of the 22-residue peptide from poliovirus serotype 1. VPg in buffer is inherently flexible, but a single conformer was obtained by adding trimethylamine N-oxide (TMAO). TMAO had only minor effects on the TOCSY spectrum. However, it increased the amount of structured peptide, as indicated by more peaks in the NOESY spectrum and an up to 300% increase in the ratio of normalized NOE cross peak intensities to that in buffer. The data for VPg in TMAO yielded a well defined structure bundle with 0.6 Å RMSD (versus 6.6 Å in buffer alone), with 10–30 unambiguous constraints per residue. The structure consists of a large loop region from residues 1 to 14, from which the reactive tyrosinate projects outward, and a C-terminal helix from residues 18 to 21 that aligns the sidechains of conserved residues on one face. The structure has a stable docking position at an area on the poliovirus polymerase crystal structure identified as a VPg binding site by mutagenesis studies. Further, UTP and ATP dock in a base-specific manner to the reactive face of VPg, held in place by residues conserved in all picornavirus VPgs.
Keywords: Viral replication; Polymerase interaction; Picornavirus; Circular dichroism; Trimethylamine N-oxide (TMAO); Solvent stabilization; Uridylylation; Post-translational modification;
Plasmodium falciparum merozoite surface protein 6 (MSP-6) derived peptides bind erythrocytes and partially inhibit parasite invasion by Ramsés López; John Valbuena; Luis E. Rodríguez; Marisol Ocampo; Ricardo Vera; Hernando Curtidor; Alvaro Puentes; Javier García; Luis E. Ramirez; Manuel E. Patarroyo (1685-1692).
This work shows that Plasmodium falciparum merozoite surface protein-6 (MSP-6) peptides specifically bind to membrane surface receptor on human erythrocytes. Three high activity binding peptides (HABPs) were found: peptides 31175 (41MYNNDKILSKNEVDTNIESN60) and 31178 (101YDIQATYQFPSTSGGNNVIP120) in the amino terminal region and 31191 (361EIDSTINNLVQEMIHLFSNNY380) at the carboxy terminal. Their binding to erythrocytes was saturable. HABPs 31191 and 31178 recognized 56 and 26 kDa receptors on erythrocyte membrane and inhibited in vitro Plasmodium falciparum merozoite invasion of erythrocytes by between 27% and 46% at 200 μg ml−1 concentration, suggesting that these MSP-6 protein peptides play a possible role in the invasion process.
Keywords: Invasion; Receptor; Ligand; Plasmodium falciparum; Vaccine; MSP-6; Binding;
Selection of peptide inhibitors against the Pseudomonas aeruginosa MurD cell wall enzyme by Catherine Paradis-Bleau; Mélanie Beaumont; Lydia Boudreault; Adrian Lloyd; François Sanschagrin; Timothy D.H. Bugg; Roger C. Levesque (1693-1700).
The purified Pseudomonas aeruginosa cell wall biosynthesis MurD amide ligase enzyme was used to screen C-7-C and 12 mers peptides from phage display libraries using competitive biopanning approaches with the specific substrates d-glutamate and ATP. From the 60 phage-encoded peptides identified, DNA was sequenced, deduced amino acid sequences aligned and two peptides were synthesized from consensus sequences identified. The UDP-N-acetylmuramyl-l-alanine MurD substrate was synthesized, purified and used to develop a spectrophotometric assay. One peptide synthesized was found to specifically inhibit ATPase activity of MurD. The IC50 value was estimated at 4 μM for the C-7-C MurDp1 peptide. The loop conformation of MurDp1 was shown to be important for the inhibition of the UDP-N-acetylmuramyl-l-alanine:d-glutamate MurD ligase. The linear 12 mers MurD2 peptide has an IC50 value of 15 mM. A conserved amino acid motif was found between MurDp2 and the bacterial glyceraldehyde 3-phosphate dehydrogenase indicating that MurDp2 binds at a protein–protein interacting site. The approach proposed and results obtained suggest that efficient peptide inhibitors as well as protein–protein interaction domains can be identified by phage display.
Keywords: Pseudomonas aeruginosa; MurD; UDP-N-acetylmuramyl-l-alanine:d-glutamate ligase; Phage display; Inhibitory peptides;
A conserved tyrosine residue of Saccharomyces cerevisiae leukotriene A4 hydrolase stabilizes the transition state of the peptidase activity by Michael W. Thompson; Erin D. Archer; Carrie E. Romer; Rebecca L. Seipelt (1701-1709).
Saccharomyces cerevisiae leukotriene A4 hydrolase (LTA4H) is a bifunctional aminopeptidase/epoxide hydrolase and a member of the M1 family of metallopeptidases. In order to obtain a more thorough understanding of the aminopeptidase activity of the enzyme, two conserved tyrosine residues, Tyr244 and Tyr456, were altered to phenylalanine and the mutant proteins characterized by determining K M and k cat for various amino acid β-naphthylamide substrates. While mutation of Tyr456 exhibited minimal effect on catalysis, mutation of Tyr244 caused an overall 25–100-fold reduction in catalytic activity for all substrates tested. Furthermore, LTA4H Y244F exhibited a 40-fold decrease in affinity for RB-3014, a transition state analog inhibitor, implicating Tyr244 in transition state stabilization.
Keywords: Aminopeptidase; Metallopeptidase; Metalloprotease; Leukotriene; Transition state; Catalysis; Mechanism; Protease; Zinc;
Quantitatively determined uptake of cell-penetrating peptides in non-mammalian cells with an evaluation of degradation and antimicrobial effects by Caroline Palm; Semharai Netzereab; Mattias Hällbrink (1710-1716).
Cell-penetrating peptides (CPPs) are carriers developed to improve mammalian cell uptake of important research tools such as antisense oligonucleotides and short interfering RNAs. However, the data on CPP uptake into non-mammalian cells are limited. We have studied the uptake and antimicrobial effects of the three representative peptides penetratin (derived from a non-mammalian protein), MAP (artificial peptide) and pVEC (derived from a mammalian protein) using fluorescence HPLC in four common model systems: insect cells (Sf9), gram-positive bacteria (Bacillus megaterium), gram-negative bacteria (Escherichia coli) and yeast (Saccharomyces cerevisiae). We demonstrate that non-mammalian cells internalize CPPs and a comparison of the uptake of the peptides show that the intracellular concentration and degradation of the peptides varies widely among organisms. In addition, these CPPs showed antimicrobial activity.
Keywords: Cell-penetrating peptides; Non-mammalian cells; Uptake; Degradation;
The antifungal protein AFP secreted by Aspergillus giganteus does not cause detrimental effects on certain mammalian cells by Henrietta Szappanos; Gyula Péter Szigeti; Balázs Pál; Zoltán Rusznák; Géza Szűcs; Éva Rajnavölgyi; József Balla; György Balla; Emőke Nagy; Éva Leiter; István Pócsi; Silke Hagen; Vera Meyer; László Csernoch (1717-1725).
The antifungal protein AFP is a small, cystein-rich protein secreted by the imperfect ascomycete Aspergillus giganteus. The protein efficiently inhibits the growth of filamentous fungi, including a variety of serious human and plant pathogens mainly of the genera Aspergillus and Fusarium, whereas AFP does not affect the growth of yeast and bacteria. This restricted susceptibility range makes it very attractive for medical or biotechnological use to combat fungal infection and contamination. We, therefore, analyzed whether AFP affects the growth or function of a number of mammalian cells. Here we show that the protein neither provokes any cytotoxic effects on human endothelial cells isolated from the umbilical vein nor activates the immune system. Moreover, potassium currents of neurons and astrocytes do not change in the presence of AFP and neither excitatory processes nor the intracellular calcium homeostasis of cultured skeletal muscle myotubes are affected by AFP. Our data, therefore, suggest that AFP is indeed a promising candidate for the therapeutic or biotechnological use as a potential antifungal agent.
Keywords: Aspergillus giganteus; Antifungal protein (AFP); Cytotoxicity; Inflammatory action; Calcium homeostasis; Potassium current;
Psc-AFP, an antifungal protein with trypsin inhibitor activity from Psoralea corylifolia seeds by Xingyong Yang; Jun Li; Xiaowen Wang; Weiguo Fang; Michael J. Bidochka; Rong She; Yuehua Xiao; Yan Pei (1726-1731).
An antifungal protein designated as Psc-AFP, with an apparent molecular mass of 18 kDa, was isolated from a traditional Chinese herb, malaytea scurfpea (Psoralea corylifolia L.). The isolation procedure entailed extraction, cation exchange chromatography on CM FF, gel filtration chromatography on Superdex 75 and reversed-phase high performance liquid chromatography on SOURCE 5RPC column. Automated Edman degradation determined the partial N-terminal sequence of Psc-AFP to be NH2-EWEPVQNGGSSYYMVPRIWA, which displayed homology with plant trypsin inhibitors. The protease inhibitor activity of Psc-AFP was then confirmed by the inhibition on trypsin. Psc-AFP at 10 μM inhibited the mycelial growth of Alternari brassicae, Aspergillus niger, Fusarium oxysporum and Rhizoctonia cerealis, suggesting that Psc-AFP has a role in the defense against pathogens.
Keywords: Psoralea corylifolia; Antifungal protein; Trypsin inhibitor; Isolation;
An antifungal protein from the pea Pisum sativum var. arvense Poir by H.X. Wang; T.B. Ng (1732-1737).
An antifungal protein with a molecular mass of 11 kDa and a lysine-rich N-terminal sequence was isolated from the seeds of the pea Pisum sativum var. arvense Poir. The antifungal protein was unadsorbed on DEAE-cellulose but adsorbed on Affi-gel blue gel and CM-cellulose. It exerted antifungal activity against Physalospora piricola with an IC50 of 0.62 μM, and also antifungal activity against Fusarium oxysporum and Mycosphaerella arachidicola. It inhibited human immunodeficiency virus type 1 reverse transcriptase with an IC50 of 4.7 μM.
Keywords: Pisum sativum; Pea; Antifungal protein; Isolation;
The Chinese bamboo leaf odorous frog (Rana (Odorrana) versabilis) and North American Rana frogs share the same families of skin antimicrobial peptides by Tianbao Chen; Mei Zhou; Pingfan Rao; Brian Walker; Chris Shaw (1738-1744).
The Chinese bamboo leaf odorous frog (Rana (Odorrana) versabilis) and the North American pickerel frog (Rana palustris) occupy different ecological niches on two different continents with no overlap in geographical distribution. R. palustris skin secretions contain a formidable array of antimicrobial peptides including homologs of brevinin-1, esculentin-1, esculentin-2, ranatuerin-2, a temporin and a family of peptides considered of unique structural attributes when isolated, palustrins 1–3. Here we describe the structures of mature peptides and precursors of eight putative antimicrobial peptides from the skin secretion of the Chinese bamboo leaf odorous frog (Rana (Odorrana) versabilis). Each peptide represents a structural homolog of respective peptide families isolated from R. palustris, including two peptides identical in primary structure to palustrin 1c and palustrin 3b. Additionally, two peptides were found to be structural homologs of ranatuerin 2B and ranatuerin 2P from the closely-related North American species, Rana berlandieri (the Rio Grande leopard frog) and Rana pipiens (the Northern leopard frog), respectively. Both palustrins and ranatuerins have hitherto been considered unique to North American ranid frogs. The use of primary structures of amphibian skin antimicrobial peptides is thus questionable as a taxonomic device or alternatively, the micro-evolution and/or ancestry of ranid frogs is more highly complex than previously thought.
Keywords: Amphibian; Peptide; Antibiotic; Taxonomy; Phylogeny; Cloning;
Molecular dissection of venom from Chinese scorpion Mesobuthus martensii: Identification and characterization of four novel disulfide-bridged venom peptides by Xian-Chun Zeng; Feng Luo; Wen-Xin Li (1745-1754).
Scorpion venom is composed of a large repertoire of biologically active polypeptides. However, most of these peptides remain to be identified and characterized. In this paper, we report the identification and characterization of four novel disulfide-bridged venom peptides (named BmKBTx, BmKITx, BmKKx1 and BmKKx2, respectively) from the Chinese scorpion, Mesobuthus martensii (also named Buthus martensii Karsch). BmKBTx is composed of 58 amino acid residues and cross-linked by three disulfide bridges. The sequence of BmKBTx shows some similarities to that of the toxin, birtoxin, and its analogs. It is likely that BmKBTx is a β-toxin active on Na+ channels, which is toxic to either insects or mammals. BmKITx is composed of 71 amino acid residues with four disulfide bridges. It is the longest venom peptide identified from M. martensii so far. BmKITx shows little sequence identity with scorpion α-toxins toxic to insects. It is likely that BmKITx is a new type of Na+-channel specific toxin active on both insects and mammals. BmKKx1 contains 38 amino acid residues cross-linked by three disulfide bridges and shows 84% sequence identity with BmTx3, an inhibitor of A-type K+ channel and HERG currents. BmKKx1 has been classified as α-KTx-15.8. BmKKx2 is composed of 36 residues and stabilized by three disulfide bridges. BmKKx2 is a new member of the γ-K+-channel toxin subfamily (classified as γ-KTx 2.2). The venoms of scorpions thus continue to provide novel toxins with potential novel actions on targets.
Keywords: Mesobuthus martensii; Buthus martensii karsch; Scorpion toxins; Na+-channel; K+-channel; cDNA library;
Isolation and characterization of a novel small cardioactive peptide-related peptide from the brain of Octopus vulgaris by Atsuhiro Kanda; Hiroyuki Minakata (1755-1761).
A novel small cardioactive peptide (SCP)-related peptide (oct-SCPRP: Ser-Asn-Gly-Tyr-Leu-Ala-Leu-Pro-Arg-Gln-NH2) was isolated from the brain of the octopus (Octopus vulgaris). cDNA encoding this precursor protein was cloned. Oct-SCPRP was shown to evoke contraction in the radula protractor muscle, and the precursor protein was highly homologous to the SCP family in the Mollusk but did not encode a related peptide, SCPB. The expression of oct-SCPRP mRNA was present not only in the peripheral nervous system (PNS) which is a motor center for the control of feeding, but also in the central nervous system (CNS) which is capable of complex analysis, learning, and controls behaviors.
Keywords: Small cardioactive peptide-related peptide; Octopus; Feeding; Cloning;
Transcriptional regulation of guanylyl cyclase/natriuretic peptide receptor-A gene by Prerna Kumar; Kiran K. Arise; Kailash N. Pandey (1762-1769).
Activation of natriuretic peptide receptor-A (NPRA) produces the second messenger cGMP, which plays a pivotal role in maintaining blood pressure and cardiovascular homeostasis. In the present study, we have examined the role of trans-acting factor Ets-1 in transcriptional regulation of Npr1 gene (coding for NPRA). Using deletional analysis of the Npr1 promoter, we have defined a 400 base pair (bp) region as the core promoter, which contains consensus binding sites for transcription factors including: Ets-1, Lyf-1, and GATA-1/2. Overexpression of Ets-1 in mouse mesangial cells (MMCs) enhanced Npr1 gene transcription by 12-fold. However, overexpression of GATA-1 or Lyf-1 repressed Npr1 basal promoter activity by 50% and 80%, respectively. The constructs having a mutant Ets-1 binding site or lacking this site failed to respond to Ets-1 activation of Npr1 gene transcription. Collectively, the present results demonstrate that Ets-1 greatly stimulates Npr1 gene promoter activity, implicating its critical role in the regulation and function of NPRA at the molecular level.
Keywords: Natriuretic peptides; Npr1 promoter; Gene transcription; Ets-1; Mesangial cells;
Role of the kallikrein–kinin system in Ang-(1-7)-induced vasodilation in mesenteric arterioles of Wistar rats studied in vivo–in situ by Rossana Anderson Marangoni; Adriana Karaoglanovic Carmona; Rita Cássia A. Tostes Passaglia; Dorothy Nigro; Zuleica Bruno Fortes; Maria Helena Catelli de Carvalho (1770-1775).
Angiotensin-(1-7) [Ang-(1-7)], exerts a variety of actions in the cardiovascular system, with an important effect being vasodilation. In this work, we investigated the relationship between the vasodilatory activity of Ang-(1-7) and the kallikrein–kinin system. Intravital microscopy was used to study the vasodilation caused by Ang-(1-7) in the mesenteric vascular bed of anesthetized Wistar rats. The topical application of Ang-(1-7) caused vasodilation of mesenteric arterioles that was reduced by A-779, JE 049 and peptidase inhibitors (aprotinin, SBTI, PKSI 527, E-64, PMSF). These results indicated that the vasodilation induced by Ang-(1-7) in the mesenteric arterioles of Wistar rats was heavily dependent on the activation of kallikrein and subsequent kinin formation.
Keywords: Angiotensin-(1-7); Bradykinin; Microcirculation; Peptidase; Kallikrein–kinin system; Renin–angiotensin system;
Neurohormones and inflammatory mediators in patients with heart failure undergoing cardiac resynchronization therapy: Time courses and prediction of response by Giuseppe Boriani; François Regoli; Davide Saporito; Cristian Martignani; Tiziano Toselli; Mauro Biffi; Gloria Francolini; Igor Diemberger; Letizia Bacchi; Claudio Rapezzi; Roberto Ferrari; Angelo Branzi (1776-1786).
Despite interest in neurohormonal activation as a determinant of prognosis in chronic heart failure (CHF) and as a target for pharmacological treatments, data are lacking on the time-related effects of electrical cardiac resynchronization therapy (CRT) on a broad spectrum of neurohormones and cytokines. The aim of this study was to assess time-courses and extents of changes within the neurohormonal profile of CHF patients treated with CRT. We performed a prospective follow-up study in 32 patients with NYHA class III–IV CHF to investigate the effects of CRT on a broad panel of neurohormones proposed for characterization of CHF patients. Levels of atrial and brain natriuretic peptides (ANP, BNP), epinephrine, norepinephrine, aldosterone, plasma renin activity, IL-6, TNF, soluble receptors sTNFR1 and 2, and chromogranin A were assessed before implantation and after 3 months of CRT; when feasible, measurements were also performed at 1 week, 1 month and 12 months (clinical evaluation, echocardiography and ECG were also performed at each time-point). The results showed that at 3 months improvement in NYHA class and echographically assessed left ventricular (LV) reverse structural remodeling were accompanied by significant reductions versus baseline in ANP and BNP, but not in other neurohormones. Moreover a baseline ANP concentration ≤150 pg/ml was a good predictor of response to CRT in terms of NYHA class reduction and reverse LV remodeling. In conclusion 3 months of CRT significantly reduce natriuretic peptides concentrations, while values of other neurohormones and inflammatory cytokines are relatively unvaried. A baseline ANP concentration ≤150 pg/ml might be a clinically useful predictor of medium-term response to CRT.
Keywords: Natriuretic peptides; Cardiac resynchronization therapy; Cytokines; Heart failure; Neurohormones; Sudden death;
Proneurotensin 1–117, a stable neurotensin precursor fragment identified in human circulation by A. Ernst; S. Hellmich; A. Bergmann (1787-1793).
Proneurotensin/neuromedin N (pro NT/NMN) is the common precursor of two biologically active peptides, neurotensin (NT) and neuromedin N (NMN). We have established antibodies against peptide sequences of the NT/NMN precursor and developed a sandwich immunoassay for the detection of pro NT/NMN immunoreactivity in human circulation. Endogenous pro NT/NMN immunoreactivity was enriched by affinity chromatography using antibodies against two different pro NT/NMN epitopes, and further purified by reversed phase HPLC. Mass spectrometry analysis revealed pro NT/NMN 1–117 as major pro NT/NMN immunoreactivity in human circulation. Pro NT/NMN 1–117 is detectable in serum from healthy individuals (n = 124; median 338.9 pmol/L). As known for NT, the release of pro NT/NMN 1–117 from the intestine into the circulation is stimulated by ingestion of an ordinary meal. Investigation of the pro NT/NMN 1–117 in vitro stability in human serum and plasma revealed that this molecule is stable for at least 48 h at room temperature. Since pro NT/NMN 1–117 is theoretically produced during precursor processing in stoichiometric amounts relative to NT and NMN, it could be a surrogate marker for the release of these bioactive peptides.
Keywords: Prohormone; Neurotensin; Neuromedin N; Pro NT/NMN 1–117; Mass spectrometry; Sandwich immunoassay; Half-life;
A hemoglobin fragment found in cervicovaginal fluid from women in labor potentiates the action of agents that promote contraction of smooth muscle cells by Amy G. Brown; Rita S. Leite; Adam J. Engler; Dennis E. Discher; Jerome F. Strauss (1794-1800).
We employed a proteomic approach to search for peptides that have a physiological role in labor. Cervicovaginal secretions were collected at term from women in labor and women at term not in labor. Samples were spotted onto weak cation exchange chips (WCX-2) and analyzed using Surface-Enhanced Laser Desorption Ionization Time-of-Flight Mass Spectrometry (SELDI-TOF-MS). Spectra were obtained for each sample and Biomarker Wizard analysis revealed 25 peaks that had significantly different peak intensity between the labor and non-laboring women. The sequences of five peaks that were significantly elevated in the labor cohort were determined using Protein Chip Interface Quadruple Time-of-Flight Mass Spectrometry (PCI-QTOF-MS). All of these peaks were identified as fragments of α or β-hemoglobin (Hb). A 2.022 kDa fragment of α-Hb (amino acids 110–128, NH2-AAHLPAEFTPAVHASLDKF-COOH) was found to potentiate smooth muscle cell contraction in response to bradykinin, oxytocin and prostaglandin-F2α. This peptide may promote vasoconstriction and augment normal labor through enhancing the action of uterotonins.
Keywords: Hemoglobin fragment; Uterotonin potentiating peptide; Smooth muscle cell contraction; Cervicovaginal secretions;
Plasma urocortin 1 in sheep: Regional sampling and effects of experimental heart failure by Christopher J. Charles; Miriam T. Rademaker; A. Mark Richards; Timothy G. Yandle (1801-1805).
Although urocortin 1 (Ucn-1) has been reported to circulate in human plasma and be raised in heart failure, little, if any, information is available regarding the source of circulating Ucn-1. Accordingly, we have performed trans-organ arteriovenous sampling for measurement of Ucn-1 concentration in anesthetized sheep before and after development of pacing-induced heart failure. Arterial plasma Ucn-1 levels measured 15.2 ± 0.5 pmol/L in normal sheep and increased significantly following development of heart failure to 19.1 ± 1.6 (p < 0.05). Small but significant positive arteriovenous gradients were observed across the hepatic and renal tissue beds in both states, with rises across the hind limb significant in normal animals and across the head in heart failure. This is the first report identifying sources of circulating Ucn-1.
Keywords: Liver; Kidney; Hind limb; Heart failure; Arteriovenous gradient;
Modifications of the human urocortin 2 peptide that improve pharmacological properties by Robert J. Isfort; Feng Wang; Michelle Tscheiner; Elizabeth Dolan; Mary Beth Bauer; Frank Lefever; Deborah Reichart; Kenneth R. Wehmeyer; Raymond A. Reilman; Bradly D. Keck; Richard T. Hinkle; Adam W. Mazur (1806-1813).
Recently, we demonstrated that the corticotropin releasing factor 2 receptor agonist, urocortin 2, demonstrated anti-atrophy effects in rodent skeletal muscle atrophy models. Compared to other CRF2R agonists however, the in vivo pharmacological potency of urocortin 2 is poor when it is administered by continuous subcutaneous infusion. Therefore, we attempted to modify the structure of urocortin 2 to improve in vivo efficacy when administered by subcutaneous infusion. By substituting amino acid residues in the linker region of urocortin 2 (residues 22–32), we have demonstrated improved in vivo potency without altering selectivity, probably through reduced CRFBP binding. In addition, attempts to shorten urocortin 2 generally resulted in inactive peptides, demonstrating that the 38 amino acid urocortin 2 peptide is the minimal pharmacophore.
Keywords: Corticotropin releasing factor; Skeletal muscle atrophy; UCN2;
Modulation of Ca2+ influx by corticotropin-releasing factor (CRF) family of peptides via CRF receptors in rat pancreatic β-cells by Kazunori Kageyama; Ryoichi Kimura; Sechiko Suga; Yoshiji Ogawa; Toshihiro Suda; Makoto Wakui (1814-1819).
The actions of the corticotropin-releasing factor (CRF) family of peptides are mediated by the seven transmembrane-domain G-protein-coupled receptors, the CRF receptors type 1 (CRF1 receptor) and type 2 (CRF2 receptor). In a previous study, we reported that CRF, an endogenous ligand for CRF1 receptor, modulated Ca2+ influx in rat pancreatic β-cells. In addition to CRF, other additional members of the family, urocortins, have been identified in mammals. Urocortin 1 (UCN 1), a peptide of the CRF family, binds both CRF1 receptor and CRF2 receptor with equal affinities. Urocortin 3 (UCN 3), a highly selective ligand for CRF2 receptor with little affinity for CRF1 receptor, has been shown in rat pancreatic β-cells. The present study focused on the effects of the CRF family peptides on intracellular Ca2+ ([Ca2+]i) concentration via CRF receptors in rat pancreatic β-cells. Microfluorimetric experiments showed that CRF (0.2 nM) and UCN 1 (0.2 nM) elevated [Ca2+]i levels. Both CRF and UCN 1 effects were attenuated by astressin, a non-selective CRF receptor antagonist. Antisauvagine-30, a selective CRF2 receptor antagonist, appeared to enhance the UCN 1 effect on the elevation of [Ca2+]i. The CRF effect on the elevation of [Ca2+]i was inhibited by the addition of UCN 3. Taken together, the activation of CRF2 receptor antagonizes the CRF1 receptor-stimulated Ca2+ influx.
Keywords: Corticotropin-releasing factor; Urocortin; Corticotropin-releasing factor receptor; β-Cells; Insulin;
Relationship between anorexigenic action of pituitary adenylate cyclase-activating polypeptide (PACAP) and that of corticotropin-releasing hormone (CRH) in the goldfish, Carassius auratus by Keisuke Maruyama; Tohru Miura; Minoru Uchiyama; Seiji Shioda; Kouhei Matsuda (1820-1826).
Our recent research has indicated that intracerebroventricular (ICV) injection of pituitary adenylate cyclase-activating polypeptide (PACAP) suppresses food intake and locomotor activity in the goldfish. However, the anorexigenic mechanism of PACAP has not yet been clarified. The aim of this study was to investigate the relationship between the anorexigenic action of PACAP and that of corticotropin-releasing hormone (CRH), which is implicated in the regulation of energy homeostasis as a powerful anorexigenic peptide in the goldfish brain. We first examined feeding-induced changes in the expression of CRH mRNA, and the effect of ICV administration of PACAP on the expression of CRH mRNA in the goldfish brain. Semiquantitative analysis revealed that the expression of CRH mRNA was significantly increased by excessive feeding for 7 days. ICV administration of PACAP at a dose sufficient to suppress food intake induced a significant increase in the expression of CRH mRNA. We also examined the effect of α-helical CRH(9–41), a CRH antagonist, on the anorexigenic action of PACAP in the goldfish. The inhibitory effect of PACAP was completely suppressed by treatment with α-helical CRH(9–41). We finally investigated the effect of ICV-administered CRH on locomotor activity in the goldfish. CRH at a dose sufficient to suppress food intake induced a significant increase in locomotor activity, unlike ICV-injected PACAP. These results suggest that, in the goldfish, the anorexigenic action of PACAP is related to the CRH neuronal pathway, but that the modulation of locomotor activity by PACAP is independent of modulation by CRH.
Keywords: PACAP; CRH; Goldfish; Anorexigenic action; CRH mRNA expression; Locomotor activity;
Functional interaction between nociceptin/orphanin FQ and α-melanocyte-stimulating hormone in the regulation of feeding by Eric M. Bomberg; Martha K. Grace; Allen S. Levine; Pawel K. Olszewski (1827-1834).
Nociceptin/orphanin FQ (N/OFQ), an endogenous agonist of the opioid N/OFQ (NOP) receptor, increases food intake when administered centrally. As N/OFQ is part of a larger neural network that governs consummatory behavior, presumably its orexigenic properties stem from interplay with other neuropeptidergic components of the feeding-related circuitry. One such peptide may be the ligand of the melanocortin-3 and -4 receptors, α-melanocyte-stimulating hormone (α-MSH), which is known to inhibit food intake. The aim of the present study was to establish whether there is a functional “interaction” between N/OFQ and α-MSH in the regulation of feeding. By using double immunostaining for c-Fos and α-MSH, we found that intracerebroventricular (i.c.v.) injection of N/OFQ at a 10 nmol dose that moderately prolongs deprivation-induced food intake in rats, decreases activation of α-MSH neurons involved in feeding termination. However, i.c.v. injections of α-MSH at doses previously established to reduce deprivation-induced feeding, do not decrease hyperphagia generated by N/OFQ in ad libitum-fed animals. Our results suggest that while α-MSH does not appear to modify the orexigenic response to N/OFQ in sated rats, the NOP receptor ligand promotes a decrease in activation of neurons synthesizing the anorexigenic peptide, α-MSH, at the time of re-feeding. Thus, to some degree, the stimulatory effect of N/OFQ on consumption may arise from this peptide's inhibitory influence on activity of anorexigenic pathways containing α-MSH.
Keywords: Nociceptin; Orphanin FQ; α-Melanocyte-stimulating hormone; Melanocortins; Feeding; c-Fos; Injection;
Proenkephalin A 119–159, a stable proenkephalin A precursor fragment identified in human circulation by A. Ernst; J. Köhrle; A. Bergmann (1835-1840).
In this report, we describe a newly developed sandwich immunoassay using antibodies against the proenkephalin A 119–159 peptide (PENK A 119–159). PENK A 119–159 immunoreactivity was detectable in the circulation of human blood donors and in cerebrospinal fluid (CSF) of patients without a neurologic disorder. The concentration was about 100 times higher in CSF than in serum. Analytical reversed phase HPLC revealed that PENK A 119–159 is the main immunoreactivity in human circulation and CSF. Moreover, PENK A 119–159 is stable in vitro for at least 48 h at room temperature as compared to the low stability of the peptides methionine- and leucine-enkephalin. This suggests the use of PENK A 119–159 measurement as surrogate molecule for the release of the mature peptides derived from proenkephalin A.
Keywords: Prohormone; PENK A 119–159; Enkephalin; Enkelytin; Sandwich-immunoassay; Half-life; CSF; Concentration gradient;
The difference in mRNA expressions of hypothalamic CCK and CCK-A and -B receptors between responder and non-responder rats to high frequency electroacupuncture analgesia by Eun-Sang Ko; Sun Kwang Kim; Jung-Taek Kim; Giseog Lee; Jae-Bok Han; Sam-Woong Rho; Moo-Chang Hong; Hyunsu Bae; Byung-Il Min (1841-1845).
The present study was performed to determine whether the expression levels of the hypothalamic cholecystokinin (CCK) and its receptors are associated with the responsiveness to high frequency electroacupuncture (EA) analgesia in rats. EA stimulation (100 Hz, 0.5 ms pulse width, 0.2–0.3 mA) was delivered to the Zusanli (ST36) acupoint of male Sprague–Dawley rats for 20 min without anesthetics or holder restraint. The analgesic effect of EA was quantified using a tail flick latency test, and subsequently animals were allocated to responder or non-responder groups. The hypothalamus of rats in each group was dissected and RNA was purified. The mRNA expressions of CCK, and CCK-A and -B receptor were determined by real-time RT-PCR. CCK mRNA levels were not significantly different in the two groups, whereas both CCK-A and -B receptors were significantly more expressed in non-responders. These results suggest that the level of CCK receptor mRNA expression in the hypothalamus, rather than CCK mRNA, has an important relationship with the individual variations to high frequency EA analgesia in rats.
Keywords: CCK; CCK-A receptor; CCK-B receptor; Acupuncture; Responder; Non-responder;
Endomorphin1 and endomorphin2, endogenous potent inhibitors of electrical field stimulation (EFS)-induced cholinergic contractions of rat isolated bronchus by Ye Yu; Yun Cui; Xiang Wang; Ying-zhe Fan; Jing Liu; Xiang Yan; Rui Wang (1846-1851).
In the present study, we determined whether endomorphin1 (EM1) and endomorphin2 (EM2), selective endogenous μ-opioid receptor (MOR) agonists, inhibited the response to EFS in rat isolated bronchus in a concentration- and frequency-dependent manner. EM1 (1 μM) produced significant inhibition at relatively low frequencies (<5 Hz) (74.02 ± 5.53%, 56.16 ± 10.24% and 37.64 ± 5.92% inhibition at 1, 2 and 4 Hz, respectively, p < 0.05 versus control), but no significant inhibition at 8, 16, 32 and 64 Hz (17.15 ± 9.4%, 14.51 ± 4.23%, 9.11 ± 2.38% and 5.93 ± 3.5%, respectively, p > 0.05 versus control). Similar modulations were observed in response to EM2 (1 μM). It is therefore considered that the inhibition effects of EM1 and EM2 may take place at frequencies under physiological conditions. Furthermore, EM1 and EM2 (0.01–10 μM) induced inhibition of cholinergic constriction in a dose-dependent manner at 1, 2 and 4 Hz. The inhibitory effect on EFS was blocked by the opioid receptor antagonist naloxone (10 μM), indicating that opioid receptors were involved. Neither EM1 nor EM2 (1 μM) had an effect on the contractile response to exogenous acetylcholine, indicating a prejunctional effect. All the results indicate that EM1 and EM2 are potent inhibitors of EFS-induced cholinergic bronchoconstriction. These also imply that EM1 and EM2 may modulate cholinergic bronchoconstriction under physiological conditions and that these tetrapeptides could have therapeutic potential in the treatment of airway diseases.
Keywords: Endomorphin1; Endomorphin2; Endogenous MOR agonists; Electrical field stimulation; Cholinergic bronchoconstriction;
Wound repair and proliferation of bronchial epithelial cells enhanced by bombesin receptor subtype 3 activation by Yu-Rong Tan; Ming-Ming Qi; Xiao-Qun Qin; Yang Xiang; Xiang Li; Yue Wang; Fei Qu; Hui-Jun Liu; Jian-Song Zhang (1852-1858).
The present study was designed to investigate the role of bombesin receptor subtype 3 (BRS-3) in airway wound repair. The results showed that: (1) There was few expression of BRS-3 mRNA in the control group. In contrast, the expression of BRS-3 mRNA was gradually increased in the early 2 days, and peaked on the fourth day, and then decreased in the ozone-stressed AHR animal. BRS-3 mRNA was distributed in the ciliated columnar epithelium, monolayer columnar epithelium cells, scattered mesenchymal cells and Type II alveolar cells; (2) The wound repair and proliferation of bronchial epithelial cells (BECs) were accelerated in a concentration-dependent manner by BRS-3 activation with P3513, which could be inhibited by PKA inhibitor H89. The study demostrated that activation of BRS-3 may play an important role in wound repair of AHR.
Keywords: Bombesin receptor subtype 3 (BRS-3); Airway hyperresponsiveness (AHR); Bronchial epithelium cells (BECs);
Signaling involved in pituitary adenylate cyclase-activating polypeptide-stimulated ADNP expression by Tomoya Nakamachi; Min Li; Seiji Shioda; Akira Arimura (1859-1864).
Activity-dependent neurotrophic protein (ADNP) was discovered as a novel response gene for VIP and has neuroprotective potential. When the VIP paralog, PACAP38 was added to mouse neuron–glia co-cultures, it induced ADNP mRNA expression in a bimodal fashion at subpico- and nanomolar concentrations with greater response at subpicomolar level. The response was attenuated by a PAC1-R antagonist at both concentrations and by a VPAC1-R antagonist at nanomolar concentration only. An IP3/PLC inhibitor attenuated the response at both concentrations of PACAP38, but a MAPK inhibitor had no effect. A PKA inhibitor suppressed the response at nanomolar concentration only. These findings suggest that ADNP expression is mediated through multiple receptors and signaling pathways that are regulated by different concentrations of PACAP.
Keywords: PACAP; ADNP; Signal transduction; Neuron/glia co-culture; Mouse;
Mutation of the phosphorylatable residue Thr429 in Glu of the human VPAC1 led to a constitutively desensitized receptor by Christelle Langlet; Ingrid Nachtergael; Patrick Robberecht; Ingrid Langer (1865-1870).
The hVPAC1 receptor is rapidly phosphorylated and internalized by agonists but not re-expressed at the membrane after washing. Mutation of Ser/Thr residues in the C-terminus reduced phosphorylation but not internalization that was abolished only when all the phosphorylatable residues were mutated. Substitution of Thr429 by Glu mimicking a phosphothreonin led to a mutant with unchanged binding properties, decreased coupling to adenylate cyclase consisting in a reduced VIP potency, increased basal and VIP stimulated phosphorylation, preserved internalization followed by a rapid receptor re-expression. These are the expected characteristics of a constitutively desensitized receptor, putting forward the role of Thr429 phosphorylation in that process.
Keywords: VPAC1 receptors; Desensitization; Receptor mutation;
Pituitary adenylate cyclase-activating peptide (PACAP) stimulates production of interleukin-6 in rat Müller cells by Masayoshi Nakatani; Tamotsu Seki; Yuko Shinohara; Chisato Taki; Shigeru Nishimura; Atsushi Takaki; Seiji Shioda (1871-1876).
Pituitary adenylate cyclase-activating peptide (PACAP) is known to regulate not only neurons but also astrocytes. Here, we investigated, both in vitro and in vivo, the effects of PACAP38 on rat Müller cells, which are the predominant glial element in the retina. Müller cells isolated from juvenile Wistar rats were treated with PACAP38 or PACAP6-38, a PACAP selective antagonist. Cell proliferation was determined by measuring the incorporation of bromodeoxyuridine with ELISA. Interleukin-6 (IL-6) levels in the culture medium were determined by a bioassay using B9 cells, IL-6 dependent hybridoma. In adult Wistar rats, the expression of IL-6 in the retina after intravitreal injection of PACAP38 (10 pmol) was assessed by immunohistochemistry. PACAP38 stimulated IL-6 production in Müller cells at a concentration as low as 10−12 M, which did not induce cell proliferation. This elevation of IL-6 production was inhibited by PACAP6-38. Radial IL-6 expression was observed throughout the retina at 2 and 3 days after PACAP38 injection. These data demonstrate that Müller cells are one of the target cells for PACAP. IL-6, which is released from Müller cells with stimulation by PACAP, may play a significant role in the retina.
Keywords: PACAP; Müller cells; Interleukin-6; Retina;
A novel approach to identifying β-secretase inhibitors: Bis-statine peptide mimetics discovered using structure and spot synthesis by Kristie Grove Bridges; Rajiv Chopra; Laura Lin; Kristine Svenson; Amy Tam; Guixian Jin; Rebecca Cowling; Frank Lovering; Tatos N. Akopian; Elizabeth DiBlasio-Smith; Bethany Annis-Freeman; Tod H. Marvell; Edward R. LaVallie; Richard S. Zollner; Jonathan Bard; William S. Somers; Mark L. Stahl; Ronald W. Kriz (1877-1885).
β-Secretase 1 (BACE1) is an aspartic protease believed to play a critical role in Alzheimer's disease. Inhibitors of this enzyme have been designed by incorporating the non-cleavable hydroxyethylene and statine isosteres into peptides corresponding to BACE1 substrate sequences. We sought to develop new methods to quickly characterize and optimize inhibitors based on the statine core. Minimal sequence requirements for binding were first established using both crystallography and peptide spot synthesis. These shortened peptide inhibitors were then optimized by using spot synthesis to perform iterative cycles of substitution and deletion. The present study resulted in the identification of novel “bis-statine” inhibitors shown by crystallography to have a unique binding mode. Our results demonstrate the application of peptide spot synthesis as an effective method for enhancing peptidomimetic drug discovery.
Keywords: Aspartic protease; Peptide inhibitor; Peptide spot synthesis; Crystallography; Bis-statine;
Molecular cloning of BNP from heart and its immunohistochemical localization in the hypothalamus of monkey by Essam Mohamed Abdelalim; Tatsuyuki Takada; Ryuzo Torii; Ikuo Tooyama (1886-1893).
Previous physiological studies have suggested central roles of brain natriuretic peptide (BNP). However, little information is available about the localization of BNP in the brain. In this study, we determined cDNA sequence encoding the entire coding region of prepro-BNP of Japanese and cynomologus monkeys, and then examined the immunohistochemical localization of BNP in the monkey hypothalamus. Japanese and cynomologus monkey prepro-BNP consisted of 132 amino acid residues with biologically active C-terminal 32 amino acids. Comparisons of deduced amino acid sequences among different species revealed high homology between monkey and human (91% in prerpro-BNP and 97% in the mature region). Immunohistochemical examination showed that BNP immunoreactive dots were observed in the paraventricular, periventricular, and supraoptic nuclei of the monkey hypothalamus. The present result suggests the central role of BNP in the neuroendocrine system in the hypothalamus.
Keywords: Natriuretic peptide; Neuroendocrine; Hypothalamus; Monkey;
Parathyroid hormone-related protein is reduced in severe chronic heart failure by George Giannakoulas; Haralambos Karvounis; George Koliakos; Thalia Damvopoulou; Theodoros Karamitsos; Christodoulos Papadopoulos; Emmanouela Dalamanga; Apostolos Hatzitolios; George Parcharidis; George Louridas (1894-1897).
In the cardiovascular system, parathyroid hormone-related peptide (PTHrP) is expressed in various cells such as cardiac vascular smooth muscle cells, coronary endothelial cells and cardiomyocytes and acts as an autocrine/paracrine substance. We compared PTHrP levels in 35 consecutive patients with severe CHF (33 male, mean age 66.2 ± 8.9 years) with 26 normal controls (24 male, mean age 63.1 ± 8.6 years). PTHrP levels were reduced in severe CHF patients (11.10 ± 1.37 fmol/ml) compared with the controls (20.62 ± 3.30 fmol/ml, p = 0.005). PTHrP values decreased as a function of New York Heart Association classification. These results suggest that PTHrP levels decrease in proportion to the severity of heart failure and could potentially be used to monitor progression of disease non-invasively.
Identification of DU 145 prostate cancer cell proteins that bind to the carboxy-terminal peptide of human PTHrP in vitro by John J. Grzesiak; Douglas W. Burton; Leonard J. Deftos; Michael Bouvet (1898-1901).
Peptides spanning the range of human parathyroid hormone-related protein (PTHrP) have been shown to bind heat shock protein-70 expressed on the surface of cancer cells with cytoprotective consequences in vitro. The present study focused on identification of intracellular proteins that interact with the carboxy-terminal peptide of human PTHrP. Using affinity chromatography, we applied extracts of DU 145 prostate cancer cells over PTHrP (140–173)–Sepharose and eluted with 8 M urea. After concentration and electrophoresis, protein bands were excised and subjected to mass spectroscopy analyses. Proteins identified included those associated with protection from oxidative stress, DNA repair, protection from apoptosis, and proteins involved in membrane trafficking and cytoskeletal rearrangement. These novel protein–protein interactions further support the hypothesis that the carboxy-terminus of PTHrP plays a role in cell survival.
Immunogenicity of xenopeptide hormone therapies by Catherine A. Schnabel; S. Edwin Fineberg; Dennis D. Kim (1902-1910).
Peptides are a growing class of agents whose therapeutic use originated with non-human treatments such as animal insulins. Xenopeptides continue to be explored for biotherapeutic development using genetic engineering, and through the rich resource of animal and plant polypeptides. One of the major concerns of therapeutic administration of xenopeptides is the potential for untoward immune responses that may lead to loss of drug efficacy or adverse events in recipients. An increased risk of immunogenicity is perceived with xenopeptides, however, human-derived therapies also induce antibody formation that in some cases has been associated with severe clinical sequelae. In this review, antibody responses to xenopeptides are highlighted looking at current hormone therapies used to treat endocrine disorders. Similar to clinical experiences with peptide-based agents in general, antibody responses against xenopeptide hormone therapies in majority of cases have been benign in nature with minimal clinical impact.
Keywords: Immunogenicity; Xenopeptide; Hormone therapies;